Enhancing Sensitivity of Detection of Immune Responses to Mycobacterium leprae Peptides in Whole-Blood Assays

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN- than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN- production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN- production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN- in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN- was mainly mediated by CD4 T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1 (MIP-1) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN--based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides. Leprosy is a disabling and stigmatizing disease caused by infection with Mycobacterium leprae. The characteristic immunological and clinical leprosy spectrum, classified by Ridley and Jopling in 1966 (25), ranges from tuberculoid (TT) or paucibacillary (PB) leprosy to lepromatous (LL) or multibacillary (MB) leprosy. In between these poles the borderline states borderline lepromatous (BL), borderline borderline (BB), and borderline tuberculoid (BT) leprosy are positioned. TT/BT patients in general show high cellular responses to M. leprae antigens injected in the skin as well as in in vitro T-cell assays; have low antibody titers to M. leprae antigens, including phenolic glycolipid I (PGL-I); and develop localized granulomatous disease with few, if any, detectable bacilli in their lesions. At the opposite end of the spectrum are LL/BL patients with a characteristic inability to generate M. leprae-specific Th1-cell responses and with disseminating progressive infection and high antibody titers to M. leprae antigens, including the M. leprae-specific cell surface antigen PGL-I. Over the last 2 decades the WHO leprosy elimination pro